Deubiquitinase USP5 regulates RIPK1 driven pyroptosis in response to myocardial ischemic reperfusion injury

Figures

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  Fig. 1. USP5 as a positive regulator for RIPK1 kinase activation and pyroptosis. (A) Caspase-1 and Caspase-8 activity was determined by an enzyme activity assay kit in CMECs. Data are expressed as mean ± SEM (n = 5), and were analyzed using two-tailed Student t test. *P < 0.05 vs. Control group. (B) Caspase-1 and Caspase-8 activity was determined by an enzyme activity assay kit in mouse serum. Data are expressed as mean ± SEM (n = 5), were analyzed using two-tailed Student t test. *P < 0.05 vs. Sham group. (C-D) Immunoblot analysis and quantification by densitometry of RIPK1 and c-GSDMD protein in HEK293T cells transfected with Myc-USP5. Data are expressed as mean ± SD (n = 3), and were analyzed using Mann-Whitney U test or two-tailed Student t test. *P < 0.05 vs. Myc-USP5 group. (E-F) Immunoblot analysis and quantification by densitometry of USP5, RIPK1, FADD and c-GSDMD protein in CMECs silenced of USP5. Data are expressed as mean ± SD (n = 3), and were analyzed using Mann-Whitney U test or two-tailed Student t test. *P < 0.05 vs. H/R group. (G-H) Immunoblot analysis and quantification by densitometry of RIPK1, Caspase-8, GSDMD and c-GSDMD in mouse CMECs from USP5+/+ or USP5+/-mice…

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  Fig. 2. USP5 interact with RIPK1. (A) Myc-tagged USP5 and Flag-tagged RIPK1were obtained by in vitro transcription and translation. Interaction between USP5 and RIPK1 was assayed by mixing USP5 and RIPK1 together followed by IP with Myc antibody and immunoblot analysis with Flag antibody. (B) Co-immunoprecipitation of endogenous USP5 with endogenous RIPK1 from CMECs under normoxia or hypoxia/reoxygenation for indicated time periods. (C) HEK293T cells expressing Myc-USP5 and Flag-RIPK1, Flag-caspase-8 or Flag-FADD were lysed. Co-immunoprecipitation of Myc-USP5 with Flag-RIPK1, Flag-caspase-8 or Flag-FADD from HEK293T cells. (D) Representative images of immunofluorescence staining of USP5 (green) and RIPK1 (red) were fixed in CMECs and incubated with a secondary antibody conjugated to Alexa Fluor 594 and Alexa Fluor 488. Colocalization between USP5 and RIPK1was examined by Confocal microscopy, Scale bar = 25 μm. (E) Schematic diagram of RIPK1and its truncation mutants. (F) Flag-tagged RIPK1or its mutants and Myc-USP5 were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with the indicated antibodies. (G)…

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  Fig. 3. USP5 deficiency promotes lysosomal degradation of RIPK1. (A-B) Immunoblot analysis and quantification by densitometry of RIPK1, Caspase-8, FADD and c-GSDMD protein or RT-PCR analysis of mouse CMECs silenced of USP5. Data are expressed as mean ± SD (n = 3), and were analyzed using Mann-Whitney U test or two-tailed Student t test. *P < 0.05 vs. USP5+/+group. (C-D) Immunoblot analysis and quantification by densitometry of RIPK1, protein or RT-PCR analysis of HCMECs silenced of USP5, then under normoxia or 2 h hypoxia/2 h reoxygenation and stimulated for TNF-α for 1 h. Data are expressed as mean ± SD (n = 3), and were analyzed using Mann-Whitney U test or two-tailed Student t test. *P < 0.05 vs. si-Control group. (E-F) Immunoblot analysis of and quantification by densitometry of RIPK1, NLRP3, Caspase-1, and caspase-4 protein from USP5+/+ or USP5+/- mouse CMECs under normoxia or 2 h hypoxia/2 h reoxygenation. Data are expressed as mean ± SD (n = 3), and were analyzed using Mann-Whitney U test or two-tailed Student t test. *P < 0.05 vs. USP5+/++normoxia group, **P < 0.05 vs. USP5+/++H/R group. (G-H) Immunoblot analysis of extracts from HCMECs, and then treated for various times with…

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  Fig. 4. USP5 reverses K63-linked ubiquitination of RIPK1. (A) Immunoblot analysis of lysates from HEK293T cells transfected with HA-tagged ubiquitin (HA-Ub), Flag-RIPK1and USP5 WT or C335A, followed by IP with anti-flag, probed with anti-HA or K63-Ub. (B) Immunoblot analysis of lysates from HEK293T cells transfected with HA-tagged K48-linked ubiquitin (K48-Ub) or HA-tagged K63-linked ubiquitin (K63-Ub), Flag-RIPK1 and Myc-USP5, followed by IP with anti-Myc, probed with anti-HA. (C) Immunoblot analysis of lysates from USP5+/+/USP5+/-mouse CMECs, followed by IP with anti-RIPK1, probed with anti-Ub, K48-Ub or K63-Ub. (D) Immunoblot analysis of extracts from HEK293T cells transfected with Flag-RIPK1and Myc-tagged USP5 or its mutants. (E) Immunoblot analysis of extracts from HCMECs transfected with USP5 wild type (WT), USP5 C335A or mutants of USP5. Similar results were obtained in three independent experiments

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  Fig. 5. RIPK1 alleviates myocardial reperfusion injury in mice. (A) Representative TTC-Evan’s Blue stained sections of heart from each group. Brick red-stained area represents the area at risk (AAR), whereas the white area indicates the infarcted size (IS). (B) Ratio of AAR/LV, IS/ARR. Data are expressed as mean ± SD (n = 10 for each), and were analyzed using Mann-Whitney U test. *P < 0.05 vs. WT + I/R group. (C) Representative cross-sectional images of the left ventricular (LV) showing no-reflow regions indicated by the absence of thioflavin-S fluorescence within the ischemic area that consists of both no-reflow (yellow arrows) and reflow zones (green arrows) following IR. (D) Ratio of no reflow area/LV. Data are expressed as mean ± SD (n = 10 for each), and were analyzed using Mann-Whitney U test. *P < 0.05 vs. WT + I/R group. (E) Survival analysis for mice subjected to 45 min ischemia and following 6 h perfusion. (*P < 0.05 vs. WT + I/R group, Kruskal Wallis test with Dunn post-hoc test for survival analysis). (F) Heart sections were obtained from WT and RIPK1flox/flox mice under sham or 45 min ischemia and following 6 h reperfusion. The sections were immunohistologically analyzed by…

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  Fig. 6. USP5 deficiency mitigates myocardial reperfusion injury in mice. (A) Representative TTC-Evan’s Blue stained sections of heart from each group. Brick red-stained area represents the area at risk (AAR), whereas the white area indicates the infarcted size (IS). (B) Ratio of AAR/LV, IS/ARR. Data are expressed as mean ± SD (n = 10 for each), and were analyzed using Mann-Whitney U test. *P < 0.05 vs. USP5+/+ +I/R group. (C) Representative cross-sectional images of the left ventricular (LV) showing no-reflow regions indicated by the absence of thioflavin-S fluorescence within the ischemic area that consists of both no-reflow (yellow arrows) and reflow zones (green arrows) following IR. (D) Ratio of no reflow area/LV. Data are expressed as mean ± SD (n = 10 for each), and were analyzed using Mann-Whitney U test. *P < 0.05 vs. USP5+/+ +I/R group. (E) Survival analysis for mice subjected to 45 min ischemia and following 6 h perfusion (*P < 0.05 vs. USP5+/+ +I/R group, Kruskal Wallis test with Dunn post-hoc test for survival analysis). (F-G) Images of echocardiography of mice and quantitative analysis of EF% and FS%. (H-I) Immunoblot analysis and quantification by densitometry of RIPK1,…

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  Fig. 7. Schematic model in which the USP5 deficiency inhibits MI/R-induced pyroptosis by suppressing RIPK1-mediated activation of caspase-8. Upon I/R, USP5 expression is upregulated, which subsequently inhibits K63-linked polyubiquitination of RIPK1 and promotes caspase-8 cleavage of gasdermin D to elicit pyroptosis. When USP5 is downregulated or mutated, RIPK1 reverse K63-linked deubiquitination, which leads to RIPK1 autophgic degradation and inhibits cell pyroptosis